Oncogenic BRAF Induces Whole-Genome Doubling Through Suppression of Cytokinesis

TLDR: Together these data suggest that common abnormalities of melanomas linked to tumorigenesis - amplified centrosomes and whole-genome doubling events - can be induced by oncogenic BRAF and other mutations that increase RAS/MAPK pathway activity.

Abstract: Melanomas and other solid tumors commonly have increased ploidy, with near-tetraploid karyotypes being most frequently observed. Such karyotypes have been shown to arise through whole-genome doubling events that occur during early stages of tumor progression. The generation of tetraploid cells via whole-genome doubling is proposed to allow nascent tumor cells the ability to sample various pro-tumorigenic genomic configurations while avoiding the negative consequences that chromosomal gains or losses have in diploid cells. Whereas a high prevalence of whole-genome doubling events has been established, the means by which whole-genome doubling arises is unclear. Here, we find that BRAFV600E, the most common mutation in melanomas, can induce whole-genome doubling via cytokinesis failure in vitro and in a zebrafish melanoma model. Mechanistically, BRAFV600E causes decreased activation and localization of RhoA, a critical cytokinesis regulator. BRAFV600E activity during G1/S phases of the cell cycle is required to suppress cytokinesis. During G1/S, BRAFV600E activity causes inappropriate centriole amplification, which is linked in part to inhibition of RhoA and suppression of cytokinesis. Together these data suggest that common abnormalities of melanomas linked to tumorigenesis – amplified centrosomes and whole-genome doubling events – can be induced by oncogenic BRAF and other mutations that increase RAS/MAPK pathway activity. Statement of Significance Whole-genome doubling is prevalent in many types of solid tumors and important in shaping tumor genomes, yet the causes of whole-genome doubling are not well understood. Here, we discover that oncogenic BRAFV600E can induce whole-genome doubling through suppression of cytokinesis, and BRAFV600E-induced whole-genome doubling can occur in melanocytes and be present in nascent melanoma cells upon tumorigenesis.

Figures

Figure 7: Progression of p53-mutant BRAFV600E-induced tetraploid cells A) Anti-Mitfa and DAPI staining of nuclei in Tg(mitfa:BRAFV600E) and Tg(mitfa:BRAFV600E);

Figure 5: Supernumerary centrosomes are observed in BRAFV600E-expressing cells A) DAPI and anti-CENTRIN-2 staining in control (-Dox) and BRAFV600E-expressing (+Dox)

Figure 2: BRAFV600E-induced binucleate, tetraploid cells arise via cytokinesis failure A) Generation of BRAFV600E-expressing RPE-1 FUCCI cell lines with a lentiviral-based doxycycline-inducible vector.

Figure 3: BRAFV600E causes cytokinesis failure by reducing the localization and function of RhoA

Figure 6: Supernumerary centrosomes contribute to RhoA downregulation and BRAFV600Einduced tetraploidy

Figure 4: BRAFV600E acts during G1/S and activates RAC1 to downregulate RhoA and generate tetraploid cells

Full text

Oncogenic BRAF Induces Whole-Genome Doubling
Through Suppression of Cytokinesis
Revati Darp
University of Massachusetts Medical School
Marc Vittoria
Boston University School of Medicine
Neil Ganem
Boston University School of Medicine https://orcid.org/0000-0003-0269-6585
Craig Ceol (  craig.ceol@umassmed.edu )
University of Massachusetts Medical School https://orcid.org/0000-0002-7188-7580
Article
Keywords: BRAF, BRAFV600E, Tetraploidy, Whole-Genome Doubling, Cytokinesis, Centriole amplication,
RhoA, Rac1
Posted Date: May 13th, 2021
DOI: https://doi.org/10.21203/rs.3.rs-455889/v1
License:   This work is licensed under a Creative Commons Attribution 4.0 International License. 
Read Full License
Version of Record: A version of this preprint was published at Nature Communications on July 15th,
2022. See the published version at https://doi.org/10.1038/s41467-022-31899-9.

1
Oncogenic BRAF Induces Whole-Genome Doubling Through Suppression of Cytokinesis
Revati Darp
1,2
, Marc A. Vittoria
3
, Neil J. Ganem
3
, Craig J. Ceol
1,2,*
1
University of Massachusetts Medical School, Program in Molecular Medicine, Worcester, MA, USA
2
University of Massachusetts Medical School, Department of Molecular, Cellular and Cancer Biology,
Worcester, MA, USA
3
Departments of Pharmacology and Experimental Therapeutics and Medicine, Division of Hematology
and Oncology, Boston University School of Medicine, Boston, MA, USA
* Corresponding author
Keywords: BRAF, BRAFV600E, Tetraploidy, Whole-Genome Doubling, Cytokinesis, Centriole
amplification, RhoA, Rac1
Running Title: Oncogenic BRAF promotes whole-genome doubling
Correspondence to:
Craig J. Ceol
email: Craig.Ceol@umassmed.edu
368 Plantation Street, ASC-1041
Worcester, MA 01695
Phone: 508-856-5509
The authors declare no potential conflicts of interest.

2
Abstract
Melanomas and other solid tumors commonly have increased ploidy, with near-tetraploid karyotypes
being most frequently observed. Such karyotypes have been shown to arise through whole-genome
doubling events that occur during early stages of tumor progression. The generation of tetraploid cells
via whole-genome doubling is proposed to allow nascent tumor cells the ability to sample various pro-
tumorigenic genomic configurations while avoiding the negative consequences that chromosomal gains
or losses have in diploid cells. Whereas a high prevalence of whole-genome doubling events has been
established, the means by which whole-genome doubling arises is unclear. Here, we find that BRAF
V600E
,
the most common mutation in melanomas, can induce whole-genome doubling via cytokinesis failure in
vitro and in a zebrafish melanoma model. Mechanistically, BRAF
V600E
causes decreased activation and
localization of RhoA, a critical cytokinesis regulator. BRAF
V600E
activity during G1/S phases of the cell
cycle is required to suppress cytokinesis. During G1/S, BRAF
V600E
activity causes inappropriate centriole
amplification, which is linked in part to inhibition of RhoA and suppression of cytokinesis. Together these
data suggest that common abnormalities of melanomas linked to tumorigenesis – amplified centrosomes
and whole-genome doubling events – can be induced by oncogenic BRAF and other mutations that
increase RAS/MAPK pathway activity.

3
Statement of Significance
Whole-genome doubling is prevalent in many types of solid tumors and important in shaping tumor
genomes, yet the causes of whole-genome doubling are not well understood. Here, we discover that
oncogenic BRAF
V600E
can induce whole-genome doubling through suppression of cytokinesis, and
BRAF
V600E
-induced whole-genome doubling can occur in melanocytes and be present in nascent
melanoma cells upon tumorigenesis.

4
Introduction
Increased ploidy is a common feature of solid tumors. The most frequently observed increased
karyotypes approach tetraploidy, which led to the hypothesis that such ‘near-tetraploid’ tumors had
undergone a whole-genome doubling (WGD) event during tumor progression and subsequently
experienced a small net loss of chromosomes
1,2
. Recent bioinformatic analyses support this hypothesis,
showing that WGD events are prevalent in a diverse set of solid tumors, and nearly 37% of all solid
tumors measured, including 40% of melanomas, experienced at least one WGD event in their
progression
3,4
. Based on these analyses, WGD frequently occurs early in tumor formation, and the
presence of tetraploid cells in some pre-cancerous lesions, such as Barrett’s esophagus and lesions of
the cervix and kidney, suggests that WGD may even precede frank tumor formation in some tissues
5-8
Tetraploidy was also observed in hyperplastic lesions of the pancreas
9
, in localized prostate cancer
10-12
and some colon adenomas
13,14
, and for certain malignancies, such as oral tumors
15
, tetraploidy is a
strong predictor of malignant transformation. Additionally, in established cancers from many tissue types
WGD is a predictor of poor clinical outcome
16
.
Tetraploidy has been experimentally linked to tumorigenesis. Viral-induced cell fusion has been
shown to enhance the transformation and tumor-forming capabilities of different cell types
17-20
.
Additionally, in mouse mammary epithelial cells that were made tetraploid through treatment with the
actin filament poison dihydrocytochalasin B, tetraploid cells were able to form tumors in mice whereas
their isogenic diploid counterparts were not
21
. In support of a role for WGD in tumorigenesis, deep
sequencing of tumor samples has shown WGD to be an early event in non-small cell lung cancer,
medulloblastoma and other tumor types
1,2,22,23
.
There are different and mutually inclusive ways in which tetraploidy could contribute to
tumorigenesis. First, tetraploidy can enable cells to become tolerant to the negative consequences of
chromosome gains, losses, gene deletions, and inactivating mutations
24-30
. Hence, tetraploidy is likely to
allow tumor cells to withstand a higher incidence of mutations, thereby increasing the probability of
adaptive changes. Second, tetraploid cells have an increased rate of chromosome missegregation
31-33
,
thus increasing the possibility that a developing tumorigenic clone will accumulate and tolerate the
mutations needed for its progression to a malignant state
34
. Thirdly, proliferating tetraploid cells are
genetically unstable and can facilitate tumor progression by giving rise to aneuploidy, a known hallmark
of cancer
35
.
Melanomas are a tumor type in which WGD is prevalent
4
. Although molecular genetic analyses
have provided great insights into the genes that are involved in melanoma, very little is known about the
process by which melanocytes with these lesions become tumorigenic, and whether any mutations
underlie WGD in tumors is unclear. We examined melanocytes in zebrafish strains that are predisposed

Frequently asked questions

Q1. What have the authors contributed in "Oncogenic braf induces whole-genome doubling through suppression of cytokinesis" ?

The most commonly observed increased karyotypes approach tetraploidy, which led to the hypothesis that such ‘ near-tetrasploid ’ tumors had undergone a whole-genome doubling ( WGD ) event during tumor progression and subsequently experienced a small net loss of chromosomes this paper. 

Q2. What is the effect of BRAFV600E on the formation of tetraploid?

If BRAFV600E acts to reduce function of RhoA, then an increase in RhoA function would be predicted to suppress the effect of BRAFV600E on the formation of binucleate, tetraploid cells. 

Q3. What was used for transfection using lipofectamine?

The lentiviral packaging plasmids pMD2.G (Addgene plasmid #12259) and psPAX2(Addgene plasmid #12260) were used for transfection using lipofectamine. 

Q4. What is the effect of BRAFV600E on melanocytes?

Nascent tumor cells in BRAFV600E-driven zebrafish melanomas are tetraploid and have higherploidyTheir data indicate that BRAFV600E can induce tetraploidy in melanocytes, and these melanocytesare prevented from progressing further by a p53-dependent block. 

Q5. How did the authors determine how BRAFV600E generates tetraploid cells?

BRAFV600E binucleate, tetraploid cells arise via failure of cytokinesisTo determine how BRAFV600E generates binucleate, tetraploid cells and to recapitulate thephenotype the authors observed in their zebrafish model, the authors developed an in vitro system suitable for mechanistic analyses. 

Q6. What is the effect of BRAFV600E on ploidy defects?

The ploidy defects are dependent on both BRAFV600E expression and p53 deficiency, mirroring the genetic synergism displayed by Tg(mitfa:BRAFV600E) and p53(lf) in melanomaformation. 

Q7. What is the role of anillin in cytokinetic processes?

Anillin localization is regulated by RhoA 46, which activates and coordinates several downstream events in the cytokinetic process. 

Q8. How did the authors determine how BRAFV600E causes cytokinesis failure?

BRAFV600E causes cytokinesis failure by reducing the localization and function of RhoATo understand how BRAFV600E inhibits cytokinesis, the authors investigated the localization and functionof proteins that are central to the cytokinetic process. 

Q9. What is the effect of aP53-dependent arrest on a newly-generated?

Newly-generated G1 binucleate tetraploid cells undergo aP53-dependent arrest, as evidenced by the progression into S phase of P53-mutant RPE-1 G1 tetraploids. 

Q10. What is the p53-dependent arrest of tetraploid cells?

To determine if a P53-dependent arrest prevents BRAFV600E-expressing tetraploids from entering the cell cycle, the authors isolatedG1 tetraploids after release from synchronization, cultured them for 24 hours, then assessed cell cycle progression. 

Q11. What is the phenotype of zebrafish melanocytes?

these zebrafish melanocytes are found as binucleates withmost nuclei appearing to have gone through a single S phase without any further mitosis. 

Q12. What is the role of RhoA in the formation of tetraploid cells?

These data indicate that BRAFV600E reduces the activity of RhoA and its downstream effector Anillin, whichunderlies the failure of cytokinesis and the formation of binucleate, tetraploid cells. 

Q13. How many nevi have a binucleate and multinucleated giant?

the presence of binucleate and multinucleated giant cells in nevi is not uncommon 81, and polyploid cells are observed at a low fraction in many nevus samples 82. 

Q14. How many centrioles per cell were observed in BRAFV600E-expressing cells?

Supernumerary Centrin-2-positive centrioles (>4 centrioles per cell) were observed in 22% of BRAFV600E-expressing cells, which is an eight-fold increase as compared to controlcells (Fig. 5B). 

Q15. What is the role of BRAFV600E in the formation of supernumerary centr?

Taken together, their data support a model in which BRAFV600E causes the formation of supernumerarycentrosomes, leading to the activation of Rac1, which in turn causes inhibition of RhoA and failure ofcytokinesis.